Polynucleotide hybridization probes offer an inexpensive, efficient and rapid means of detecting, localizing and isolating "target" nucleotide sequences for clinical and research purposes. Klausner et al., Biotechnology, August 1983:471-478, provide interesting background on polynucleotide hybridization probes, including discussion of preparation and use.
Know methods for preparing polynucleotide hybridization probes and for using such probes are well documented in the literature. See, for example, Southern, J. Mol. Biol. 98:503-517 (1975); Falkow et al., U.S. Pat. No. 4,385,535; Leary et al., Proc. Natl. Acad. Sci. 80:4045-4049 (1983); Langer-Safer et al., Proc. Natl. Acad. Sci. 79:4381-4385 (1982); and Langer et al., Proc. Natl. Acad. Sci. 78:6633-6637 (1981); and Kohne, WO84-02721, published July 19, 1984.
As disclosed by these and other references, such known methods for preparing probes typically comprise cloning a probe region into a double stranded DNA plasmid. The plasmid carrying the probe region is labeled, typically by enzymatic polymerization techniques. Such techniques include, for example, nick translation. (Rigby et al., J. Mol Biol. 113:237 (1977)); gap-filling (Bourguignon et al., J. Virol. 20:290 (1976)); and terminal addition, which techniques are carried out in the presence of modified nucleotide triphosphates. Chemical modification techniques, including those reported in references cited below, can also be used.
Brigati et al., Virology 126:32-50 (1983), report that biotin-labeled probes in which biotin is bound to deoxyuridine triphosphate (dUTP) by a long chain (11 or 16 atoms) are advantageous over probes in which the biotin label is bound to the nucleotides by a short chain (4 atoms). The polynucleotide probes are labeled by enzymatic incorporation of biotinylated dUTP by nick translation, using DNA polymerase I.
Ward et al., EP-A-63,879, disclose that label molecules are preferably not bound to positions in a nucleotide ring which would cause interference with hydrogen bonding by a probe to a target sequence. The authors disclose, as a solution to this problem, binding label molecules at the 5-positions of pyrimidines (thymine, cytosine and uracil), at the 7-positions of purines (adenine and guanine), and at the 7-positions of deazapurines. As a further solution, Ward et al. disclose binding label molecules to nucleotides with an extended linker arm, as per Brigati et al., above.
Kourilsky et al., GB No. 2,019,408, disclose chemical modification, by the cytochrome C-biotin technique reported by Manning et al., Biochem. 16:1364 (1977), of polynucleotide hybridization probes and labeling of such modified probes prior or subsequent to hybridization to a sample. Chemical labeling methods are also, disclosed by Renz et al., Nucl. Acids Res. 12:3435 (1984) and Tchen et al., Proc. Nat'l. Acad. Sci. USA 81:3466 (1984). The Renz procedure couples enzymes directly to probes by methods similar to those of Kourilsky et al., GB No. 2,019,408 and Manning et al., cited above. In the Tchen procedure, modification of greater than 5 to 10% of the nucleotide residues adversely affects probe hybridization.